Characterization of human aldehyde oxidase: substrate specificities, mode of inhibition and superoxide production

人醛氧化酶的表征：底物特异性、抑制模式和超氧化物产生

• 批准号: 224728554
• 负责人: Professorin Dr. Silke Leimkühler
• 金额: --
• 依托单位: Arbeitsgruppe für Molekulare Enzymologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/224728554?language=en
• 关键词: Characterization; human; aldehyde; oxidase; substrate

Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. So far, the physiological function of AOX for mammals is unknown. However, human AOX1 is of increasing pharmacological interest due to its recognized role in phase I drug metabolism. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. We established a codon-optimized heterologous expression system for human AOX1 in Escherichia coli. This expression system now enables us to obtain sufficient amounts of active protein for kinetic characterizations and sets the basis for site-directed mutagenesis and structure-function studies. To define the physiological function of human AOX1 the active site will be investigated in detail. We plan to characterize selected single polynucleotide polymorphisms (SNPs) identified around the substrate and inhibitor binding site in human individuals and analyze their effect on hAOX1 activity. We will further introduce amino acid exchanges around the FAD site and will analyze the effect on intramolecular electron transfer. Further, we will analyze the role of hAOX1 in the production of reactive oxygen species (ROS), since hAOX1 as a true oxidase was suggested to significantly contribute to the production of high amounts of endogenous O2-. The co-crystal structures of hAOX1 with substrates and inhibitors will be solved and will help to understand the mechanism of substrate and inhibitor binding for future drug developments. Our studies will be particularly important to define the physiopathological functions of AOX1 and will help to understand the drug metabolizing role of AOX1 for humans in general.

哺乳动物醛氧化酶（AOX）是钼氟酶，在包括人类和啮齿动物在内的各种哺乳动物物种中存在于许多组织中。不同的物种包含不同数量的AOX同工型。到目前为止，哺乳动物对AOX的生理功能尚不清楚。然而，由于人类在I期药物代谢中的认可作用，人类AOX1具有增加药理学的兴趣。特别是，除人以外的哺乳动物表达多种组织特异性AOX酶的原因是未知的。我们在大肠杆菌中建立了人类AOX1的密码子优化异源表达系统。现在，此表达系统使我们能够获得足够数量的活性蛋白来进行动力学特征，并为位置定向的诱变和结构功能研究设定了基础。为了定义人AOX1的生理功能，将详细研究主动位点。我们计划表征人个体围绕底物和抑制剂结合位点鉴定的选定的单核苷酸多态性（SNP），并分析其对HAOX1活性的影响。我们将进一步引入FAD位点周围的氨基酸交换，并将分析对分子内电子转移的影响。此外，我们将分析HAOX1在活性氧（ROS）产生中的作用，因为HAOX1作为一种真正的氧化酶有明显的促进大量内源性O2-的产生。 HAOX1与底物和抑制剂的共结构结构将得到解决，并将有助于了解底物和抑制剂结合的机理，以实现未来的药物开发。我们的研究对于定义AOX1的生理病理学功能将特别重要，并将有助于了解AOX1对人类的药物代谢作用。